Processes and materials for carrying out microchemical and microbiological tests

ABSTRACT

A microbiological culture test process for detecting the presence of nitrate-reducing microorganisms in an inoculum sample, which comprises 
     (a) culturing a microorganism to be tested in the presence of adequately nitrite-free nitrate, for a period sufficient to allow reduction of nitrate by any microorganism (if present) that has the capacity to reduce nitrate to nitrite, (e.g. 1-6 hours); 
     (b) exposing the culture medium after culture to (diazotizing) acid conditions in the presence of an amino-containing fluorophor thereby to cause diazotization of the fluorophor to the extent of any nitrite present in the medium to form a diazonium derivative (or further reaction product thereof) which is substantially less fluorescent (or fluoresces at a substantially different wavelength) than said amino-containing fluorophor, and 
     (c) assessing the fluorescence of the culture medium as treated by step (b), thereby to show a reduction in fluorescence in the presence of a nitrate-reducing microorganism.

This invention relates to processes and materials for carrying outmicrochemical and microbiological tests. In particular, it relates tochemical tests to detect the presence of small quantities of nitrite,and the application of such tests to detecting the production of nitriteby microorganisms in culture, e.g. by reduction of nitrate supplied inthe culture medium.

A well established test for small quantities of nitrite is known as theGriess test. This test relies on treating an aqueous material suspectedof containing nitrate with p-aminobenzenesulphonic acid in strong acidsolution (e.g. 5 N acetic acid) and with alpha-dimethylaminoaphthalene,also in strong acid solution.

In the presence of nitrite the test develops a red dye colour, but nocolour is given if the test is carried out with a nitrite-free sample.

This test is widely applied as part of a technique for the speciesidentification and classification of bacteria by their culturalapplication in many clinical microbiology laboratories. The test canalso be used to detect traces of nitrite in other samples, e.g. watersamples to be analysed in public health laboratories.

As it stands, this test generally requires visual assessment of colourdevelopment, and it involves the use and handling of two aromatic aminereagents which are somewhat noxious and chemically closely related toknown carcinogens.

The aim of this invention is to provide a nitrite test that can beassessed by fluorescence in the resulting test material. It is also theaim of the invention to provide tests that can give a fluorescent resultin a form suited to automatic fluorometric assessment, especially forexample by fluorometry that uses similar excitation and emissionwavelengths as other and possibly hitherto chemically unrelatedmicrobial tests, which can therefore be assessed simultaneously or inthe same batch by similar or the same instrumentation. A further aim ofthe invention is to provide nitrite detection tests adapted tomicrobiological use for assessment of the nitrate-reducing capacity ofmicrobial cultures, e.g. to assist in their identification, in which theuse of noxious reagents to develop a colour result is minimised oravoided.

According to the present invention, nitrite is detected in a samplepossibly containing small quantities of nitrite, especially for examplean aqueous sample, such as for example a suspension or culture ofmicroorganisms cultured in the presence of adequately nitrite-freenitrate for a period that allows reduction of nitrate, if possible, or aspecimen of groundwater, by contacting the sample under acid conditionswith a small quanity of an acid-stable fluorophor which has a content ofamino groups, and in which the amino groups contribute substantially tothe fluorescence, thereby to cause reaction between the amino groups ofthe fluorophor and any nitrite present under the acid conditions, andassessing the presence or quantity of nitrite by change in thefluorescence of the fluorophor as a result of the reaction.

A microbiological culture test process according to the invention, fordetecting the presence of nitrate-reducing microorganisms is an inoculumsample, which comprises:

(a) culturing a microorganism to be tested in the presence of adequatelynitrite-free nitrate, for a period sufficient to allow reduction ofnitrate by any microorganism (if present) that has the capacity toreduce nitrate to nitrite (e.g. 1-6 hours);

(b) exposing the culture medium after culture to (diazotising) acidconditions in the presence of an amino-containing fluorophor thereby tocause diazotisation of the fluorophor to the extent of any nitritepresent in the medium to form a diazonium derivative (or furtherreaction product thereof) which is substantially less fluorescent (orfluoresces at a substantially different wavelength) than saidamino-containing fluorophor, and

(c) assessing the fluorescence of the culture medium as treated by step(b), thereby to show a reduction in fluorescence in the presence of anitrate-reducing microorganism.

Preferably the fluorophor is one which itself fluoresces under acidconditions but of which the acid reaction product with nitrite issubstantially non-fluorescent under acid conditions, and thefluorescence is assessed or measured while the product is still underthe acid conditions.

A suitable example of a fluorophor which can be used for this inventionis an amino derivative of coumarin or flavone, for example a 7-aminocoumarin (or a 3-amino-flavone), especially for example 7-amino-4-methylcoumarin (hereafter referred to as MCA).

It is believed that the test exploits the difference in fluorescencebetween the added amine reagent and its corresponding diazoniumderivative formed during the test in the presence of nitrite under acidconditions.

The diazotising acid conditions can be achieved for example byacidifying the aqueous mixture of the sample to be tested and thefluorophor up to 2N-3N concentration of e.g. a strong or moderatelystrong acid, such as hydrochloric acid or acetic acid.

In a microbiological test for nitrate reduction, using theabove-described method for detecting nitrite, suitable conditions arefor example as follows.

EXAMPLE

A microbial inoculum is prepared containing about 10⁷ organisms per ml(final concentration) and the following further ingredients in the finalconcentrations noted:

Oxoid nutrient broth No. 2 (Trade Mark): 25g/l

Potassium nitrate (nitrite-free): 1g/l

Sodium chloride: 8.5g/l

MCA (7-amino-4-methyl coumarin): 15 μM, (equivalent to 2.64 mg/l).

It is convenient to handle a standardised quantity of 100 μl in eachtest sample.

Of course, the other materials present in the culture broth, and anyother reagents, are chosen from those that do not already lead toquenching of the relevant fluorescence irrespective of the progress ofthe diazotising reaction.

This inoculum and broth-reagent mixture is incubated at 37° C. for aperiod within the range of about 1 hour to about 6 hours, and thenacidified using a drop of 5N acetic acid solution.

After a short further period, the fluorescence of a test mixture can becompared with the fluorescence of control mixtures, and any substantialdiminution in fluorescence can be taken to indicate the presence ofnitrite produced by the microorganism by reduction of nitrate.

If the test mixture is incubated for much longer than 6 hours, e.g.overnight for about 18 hours, then it is possible for certainnitrate-reducing microbes to denitrify the nitrite further to nitrogen.In such a case the test will not give a diminution in fluorescence toindicate the presence of nitrite. However, as with the traditional test,these denitrifying organisms can be distinguished from the nitratenon-reducers by adding to the acid test mixture, after assessment hasshown a lack of nitrate, a reducing agent that will itself reducenitrate to nitrite (such as zinc dust under acid conditions). Residualnitrate will then, if present, be reduced to nitrite and give thecharacteristic diminution of fluorescence. Failure of the test to show adiminution of fluorescence even after addition of the external reducingagent indicates consumption of both the nitrate and any nitrite producedfrom it, i.e. by the action of denitrifying organisms.

However, the preferred method of carrying out the tests to incubate theinoculum mixture only for about 1-6 hours, during which shorter periodswe find that even denitrifying organisms do not eliminate the nitrite sofar produced: under these conditions both denitrifying andnon-denitrifying nitrate-reducers give similar nitrite-positive results.

It is considered to be advisable, in carrying out several embodiments ofthe test herein described, to avoid realkalizing the acid test mixturebefore measuring or assessing its fluorescence, to avoid the possibledevelopment of other interfering sources of fluorescence in thismixtures.

It can be seen from the above description that the invention provides amethod of nitrite detection or estimation and a method of detecting theproperty of nitrate reduction in microorganisms: the invention alsocomprises materials for carrying out such tests. In particular, thematerials can comprise a microbiological broth containing (besides usualmicrobial culture medium ingredients) (adequately nitrite-free) nitrateand the amino-containing fluorophor in suitable quantities (for examplethe quantities given in detail above), to be used in association with anacidifying reagent, such as the examples given above. If desired, aconvenient embodiment can be used in which the indicated brothconstituents, (or a selection of them including the essential nitrate,and optionally also the fluorophor), can be presented in dry form in oneor more wells of a prepared (usually sterile) microtitre tray for use incarrying out microbial culture tests (usually protected by a removableadhesive sealing cover). An alternative dry form presents some or all ofthe materials in or associated with (usually sterile) containers forpreparing a microbial inoculum suspension, e.g. a bottle or othercontainer with a dry preparation of the materials contained therein; ora closure for such a bottle, such as a screw cap, with a dry preparationof the materials carried on a surface which in use is to contact theinoculum so that the materials may be dispersed therethrough, such asthe inner surface of the screw cap or the surface of a wad placed withinthe screw cap; or in a dry form on a carrier material suitable to beadded to such an inoculum or to a culture well, so that the materialsare dispersed through the inoculum or culture suspension, such as stripsor discs of paper or other cellulosic or non-cellulosic, fibrous ornon-fibrous carrier sheet material.

All such preparations are desirably maintained dry and sterile beforeuse, e.g. sealed within sterile foil sealing or packaging material.

One of the special advantages of the processes described herein is thatthe nitrate reduction test gives a fluoroscopic/fluorometric result thatcan be measured optically with the same excitation/emission wavelengthsas a number of other (known in themselves) microbial culture tests (e.g.in the presence of various quantities or types of antimicrobial) givinga variably fluorescent result: e.g. culture tests in which a fluorogenicsubstrate such as 4-methyl umbelliferone phosphate or other ester and/oran aminoacyl or peptidyl 7-amino-4-methyl coumarin is hydrolysed tofluorophor during growth of the microorganism if this growth occurs.

Accordingly, combination tests including cultures for nitrate reductiontesting and such antimicrobial culture tests are also included withinthe scope of the invention.

Other modifications and variations within the scope of the describedinvention will be apparent to the skilled reader as a result of readingthe above disclosure, and the several features described by way ofexample can be presented in any desired combinations.

I claim:
 1. A microbiological culture test process for detecting thepresence of nitrate-reducing microorganisms in an inoculum sample, whichcomprises(a) culturing a microorganism to be tested in the presence ofsubstantially nitrate-free nitrate and of a diazotizableamino-containing fluorophor selected from the group consisting ofcoumarins and flavones, for a period sufficient to allow reduction ofnitrate by said microorganism having the capacity to reduce nitrate tonitrite,; (b) exposing the culture medium after culture to diazotizingacid conditions thereby to cause diazotization of the fluorophor to theextent of any nitrite present in the medium to form a diazoniumderivative which is substantially less fluorescent at a predeterminedwavelength at which said fluorophor is fluorescent than saidamino-containing fluorophor when assessed for fluorescence under saidacid conditions, and (c) assessing the fluorescence of the culturemedium as treated by step (b) at said predetermined wavelength under theacid conditions established in step (b), thereby to detect presence of anitrate-reducing microorganism by showing a reduction in fluorescence.2. A process according to claim 1 in which the fluorophor is afluorescent amino-coumarin.
 3. A process according to claim 2, in whichthe fluorophor is 7-amino-methyl coumarin.
 4. A process according toclaim 1, wherein the microorganism is cultured in the presence ofnitrite-free potassium nitrate.
 5. A process according to claim 1,wherein the acid conditions of step (b) are produced by adding acid tothe culture to give an acid concentration in the range about 2N-3N.
 6. Aprocess according to claim 5 in which the acid is hydrochloric acid oracetic acid.
 7. A process according to claim 1 in which the culturingstep (a) is conducted for a period of 1 to 6 hours.
 8. A processaccording to claim 7 in which the phosphor is a fluorescentamino-coumarin, the microorganism is cultured in the presence ofnitrate-free potassium nitrate and the acid conditions of step (b) areproduced by adding acid to the culture to give an acid concentration inthe range of about 2N-3N.
 9. A process according to claim 8 in which thefluorophor is 7-amino-4-methyl coumarin and the acid is hydrochloricacid or acetic acid.
 10. A microbial culture test medium for detectingnitrate-reducing microorganisms, comprising microbial culture mediumwhich is substantially nitrite-free, a nitrate source, and adiazotisable amino-group-containing fluorophor selected from the groupconsisting of coumarins and flavones, wherein said fluorophor yieldsreduced fluorescence in the diazotised state when measured at apredetermined wavelength under diazotising acid conditions.
 11. Amicrobial culture test medium according to claim 10, wherein saidfluorophor is a fluorescent amino-coumarin.
 12. A microbial culture testmedium according to claim 11, wherein said fluorophor is7-amino-4-methyl coumarin.
 13. A test kit for detecting nitrate-reducingmicroorganisms,comprising a prepared test tray with a plurality ofreceptacles, wherein at least one receptacle contains microbialinoculation medium, and, in dry form, nitrite-free nitrate, anddiazotisable amino-group-containing fluorophor selected from the groupconsisting of coumarins and flavones which yield reduced fluorescence inthe diazotised state when measured at a predetermined wavelength underdiazotising acid conditions.